Delivery of functional non-coding RNAs (ncRNA) such as ribozyme, microRNA (miRNA), short hairpin RNA (shRNA) and small interfering RNA (siRNA) is the major obstacle hindering the development of RNA interference (RNAi)-based therapy in vivo. Low penetration and high degradation of these ncRNAs are the problems of delivery. In order to overcome these problems, functional ncRNAs must be protected by a delivery agent that is able to not only preserve their structural integrity but also facilitate their uptake by cells in vivo. Yet, none of previously found liposomal or sugar-based delivery agents can fulfill both needs.
Nucleic acid compositions like ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) are negatively charged molecules and hence tend to attract positively charged materials. On the other hand, the cell membrane consists of phospholipid bilayer, which contains abundant fatty acids and thus is also negatively charged. As a result, naked RNA/DNA will be repelled by the cell membrane and cannot be directly delivered into cells. In order to overcome this problem, one preferred traditional delivery method is liposomal transfection using lipid-based liposomes to encapsulate DNAs/RNAs for intracellular delivery. The mechanism of liposomal transfection is achieved by fusion of liposomes to the phospholipid bilayer of the cell membrane, resulting in passive diffusion of the liposome-encapsulated RNAs/DNAs into the cells. To further improve their delivery efficiency, those liposome molecules are often modified by adding long carbon chains (i.e. glycolipids) or positively charged chemical groups, or both, such as polyethylene glycol [PEG; H—(O—CH2—CH2)n—OH] (Immordino et al, 2006), glycerol esters (WO2011143237 to Meyering), glycerol monooleate (Pereira et al, 2002; Zhen et al, 2012), and aminated/amino poly(glycerol methacrylate)s (Gao et al, 2010 and 2011). However, due to their limit by passive diffusion, the efficiency of these liposomal methods is generally not comparable to an active delivery method based on endocytosis.
In a liposomal delivery system, glycerol is often used as a polymer linker to connect the long carbon chains of fatty acids and phospholipids, such as monooleate and glycerol esters. Modifications in these long carbon chains can form charged chemical groups to interact with DNA/RNA; yet, those charged carbon chains do not possess any ability (i.e. polarity) to protect DNA/RNA from degradation. Alternatively, glycerol also can serve as a side chain in a delivery polymer, such as aminated/amino poly(glycerol methacrylate)s. Those aminated glycerol side chains in such acrylate polymers carry positively charged groups that can form hydrogen bonding (H-bond) with DNA and RNA (Gao et al, 2010). Nevertheless, it is known that the duplex and hairpin structures of DNA and RNA are also formed and maintained by H-bonds. As a result, the H-bonds formed by aminated/amino poly(glycerol methacrylate)s will disturb the structural integrity of DNA/RNA duplexes and hairpins, which are actually required for maintaining the function of many currently known nucleic acid-based drug agents, such as miRNA, shRNA, siRNA, ribozyme and DNA vaccine. Conceivably, none of these liposomal delivery systems can protect DNA and RNA from degradation.
Sugar-based delivery is another preferred transfection method, which is designed to improve the low efficiency of liposomal delivery. Sugar-encapsulated DNAs/RNAs can be absorbed by cells via an active endocytosis mechanism, which increases their concentration in cells and hence their functional efficacy as well. A variety of compositions have been used in these sugar-based delivery systems, including sugar-based surfactants (EP0535534 to Nair; WO2009029046 to Kim), poly(sugar acrylate) ploymers (U.S. Pat. No. 5,618,933 to Dordick), sugar-grafted liposomes (Banerjee et al, 1996), lipid-protein-sugar particles (WO2002032398 to Kohane et al), poly(glycosylated amino acid)s (Davis et al, 2002), lipoamino acid-/glycopeptide- and/or liposaccharide-conjugants (Blanchfield et al, 2004), pectin/chitosan/lecithin nanoparticles (Morris et al, 2010; Cuna et al, 2006; Graf et al, 2008), sugar-PEG-based polymers (Davis et al, 2010; Bhatia et al, 2011), and boron-saccharide complexes (Ellis et al, 2012). However, none of these sugar-based compositions have been reported to protect the structural integrity of RNA and DNA from degradation. Also, because polysaccharides and sugars do not normally carry any charge, many of these methods still need to be used in conjunction with liposomes in order to encapsulate DNAs/RNAs. As a result, difficult formulation is another problem.
In sum, there is currently no delivery agent that can efficiently deliver RNAs/DNAs into cells while protecting their intact strand structures, particularly duplexes and hairpins, during delivery. Moreover, since these previously developed delivery agents can not been found in any living biological system and have not been tested for in-vivo delivery yet, their safety and in-vivo efficiency are highly uncertain. Therefore, it is highly desirable to have a novel delivery system that not only safely exists in a living system but also is useful for efficiently deliver RNAs/DNAs into cells in vitro as well as in vivo while protecting their intact strand structures, in particular duplexes and hairpins.